Mycoplasmas are a group of pathogenic microorganisms of the Class Mollicutes characterized by having a small size and lacking a cell wall. These microorganisms are among the smallest-known organisms capable of a free living existence, and are important pathogens in man, plants and animals. For example, atypical pneumonia and non-gonococcal urethritis are common mycoplasma infections in man. Mycoplasmas have also been associated with rheumatoid arthritis, spontaneous abortion, infertility and other genital tract diseases, and certain autoimmune disease states. Moreover, mycoplasmas are common contaminants in cell cultures. In biological research, mycoplasma contamination of tissue culture is a serious problem which demands constant monitoring.
Not surprisingly, these organisms are extremely fastidious and at present there are no cost-effective specific diagnostic procedures to determine the presence of mycoplasma infections. The most commonly employed detection methods for mycoplasmas in clinical samples are serological and cultural. The serological methods are subject to false positives and the cultural methods are costly, time consuming and tedious. Many of the biochemical techniques in current usage for detection of microbial contaminants in cell cultures do not specifically detect mycoplasmas but rather indicate the presence of any prokaryote or simple eukaryote such as yeast and fungi, and some may even detect viruses. Such a test is advantageous if one is interested only in the knowledge that a microbial agent is present, but if one is searching for a suspected etiological agent of an animal or human disease it is obviously necessary to classify the agent as fully as possible.
Further, the above procedures are hampered by special problems. For example, there are apparently "non-cultivable" mycoplasmas which are not detected by conventional culture methods. In addition, in the case of immunofluorescence tests more than one antibody might be required to identify the particular organism since more than nine different mycoplasma species are common tissue culture contaminants. Also, DNA stains are not necessarily mycoplasma-specific.
Therefore, a simple, sensitive, specific, cost-effective, and rapid mycoplasma detection system has been a desideratum in the fields of diagnostic medicine and biological research.
The use of nucleotide sequence homology and nucleic acid hybridization kinetics has become a widely-employed technique for detecting various organisms in cells and cell cultures. However, prior to this invention reliable and specific DNA probes have not been available for mycoplasma detection.
The present invention proceeds by the use of specific mycoplasma ribosomal RNA gene fragments which are labeled or tagged by a variety of techniques, such as radioisotope labeling, biotin labeling, PEI-peroxidase conjugates, or fluorescent antibody tagging ELISA methods, for the specific and sensitive detection of mycoplasmas in clinical specimens, cells or cell cultures by DNA or RNA hybridization.
In one aspect of the invention, a DNA sequence from the 16S RNA gene of mycoplasma is provided, which includes a nucleotide sequence selected from the group consisting of AACACGTATC, CGAATCAGCTATGTCG, GAGGTT----AAC, ATCCGGATTTATT, TCTCAGTTCGGATTGA, AGGTGGTGCATGGTTG, TCCTGGCTCAGGAT, ATACATAGGT, MCTATGTGC, AATTTTTCACAATG, TCTCGGGTCT, and TAGATATATG which code for mycoplasma ribosomal RNA (rRNA) where T represents thymine, G represent guanine, A represents adenine, C represents cytosine and--indicates a nucleotide deletion within the sequence with respect to the comparable sequence in E. coli. These fragments differ significantly from the 16S RNA gene of E. coli, and thus form the basis for mycoplasma-specific probes which are constituted of labeled nucleotide sequences complementary to the above.
In another aspect of the invention, identified DNA sequences of a 16S RNA gene are provided which include nucleotide sequences selected from the group consisting of ACGGGTGAGT, TMTACCGCAT, TACGGGAGGCAGCAGT, GTGGGGAGCAAA, AGGATTAGATACCCT, CCGTAAACGAT, GAATTGACGGGG, CCCGCACAAG, GGTGGAGCATGT, TGTTGGGTTAAGTCCCGCAACGA, GGGATGACGT, ACGTGCTACAATG, CTAGTAATCG, TGTACACACCGCCCGTCA, AAGTCGTAACAAGGTA, and TGGATCACCTCCTT, which code for prokaryotic rRNA. These fragments represent regions within the 16S RNA gene that are identical for E. coli and all mycoplasmas examined. Universal probes for all prokaryotes are constituted of labeled nucleotide sequences complementary to these fragments.
In general the invention comprises a method for determining the presence of a prokaryotic organism which contains a nucleic acid including a particular nucleotide sequence which is present in nucleic acids from prokaryotic organisms but absent in nucleic acids from eukaryotic organisms, which comprises contacting a medium which may contain a nucleic acid or nucleic acid fragment from said prokaryotic organism including said particular nucleotide sequence with an oligonucleotide, including a nucleotide sequence complementary to said particular nucleotide sequence, whereby said oligonucleotide hybridizes with any nucleic acid or nucleic acid fragment from said prokaryotic organism, including said particular nucleotide sequence which may be present in said medium, and detecting the presence of any nucleic acid or nucleic acid fragment hybridized with said oligonucleotide.
Other aspects of the invention concern the specific biological probes used for detecting mycoplasmas or prokaryotes in general in accordance with the above described process and the identification and production of such probes.